Fig. 3. L-EV treatment in CRST-treated mice reversed stress-induced decreased expression of BDNF in the hippocampus and rescued stress-induced depressive-like behaviors. (A) Experimental design (Exp.2). Mice exposed to CRST (2h×14 d RST) were treated with saline, imipramine (20 mg/kg), and L-EV (0.1 µg/kg for days 1~5; 0.18 µg/kg for days 6~7; 0.27 µg/kg for day 8 and thereafter). Behavior tests were performed on post-stress days 14~16 (p14~p16; behavior tests, #1) and post-stress days 28~30 (p28~p30; behavior tests, #2). Mice were sacrificed on post-stress day 35 (p35). Control mice injected with saline (CON+veh), mice treated with L-EV (CON+L-EV), mice treated with repeated restraint and injected with saline (CRST+veh), and mice treated with repeated restraint and injected with L-EV (CRST+L-EV) and mice treated with repeated restraint and injected with imipramine (CRST+IMI) were prepared. (B) Body weight changes of CON+veh, CON+L-EV, CRST+veh, CRST+L-EV, and CRST+IMI (n=8~12 animals per group). (C) Expression levels of Ngf, tBdnf, Bdnf1, Bdnf4, Nt3, Nt4/5, and Trkb in the hippocampus of CON+veh, CON+L-EV, CRST+veh, CRST+L-EV, and CRST+IMI (n=6~8 animals and 4~6 PCR repeats per group). For the comparison of CON+veh, CON+L-EV, CRST+veh, and CRST+L-EV groups, two-way ANOVA followed by Bonferroni post hoc test was used (Ngf, F(1,14)=1.549 and p=0.2337 for CRST main effect; F(1,14)=0.3197 and p=0.5808 for L-EV main effect; F(1,14)=0.1826 and p=0.6757 for CRST×L-EV; tBdnf, F(1,14)=4.871 and p=0.0445 for CRST main effect; F(1,14)=4.274 and p=0.0577 for L-EV main effect; F(1,14)=4.752 and p=0.0468 for CRST×L-EV; Bdnf1, F(1,12)=0.5907 and p=0.4570 for CRST main effect; F(1,12)=6.682 and p=0.0239 for L-EV main effect; F(1,12)=8.992 and p=0.0111 for CRST×L-EV; Bdnf4, F(1,13)=25.35 and p=0.0002 for CRST main effect; F(1,13)=7.794 and p=0.0153 for L-EV main effect; F(1,13)=17.60 and p=0.0010 for CRST×L-EV; Nt3, F(1,14)=43.45 and p<0.0001 for CRST main effect; F(1,14)=3.274 and p=0.0919 for L-EV main effect; F(1,14)=22.21 and p=0.0003 for CRST×L-EV; Nt4/5, F(1,11)=0.0645 and p=0.8042 for CRST main effect; F(1,11)=6.108 and p=0.0310 for L-EV main effect; F(1,11)=15.76 and p=0.0022 for CRST×L-EV; Trkb, F(1,14)=0.3743 and p=0.5505 for CRST main effect; F(1,14)=7.106 and p=0.0185 for L-EV main effect; F(1,14)=1.669 and p=0.2173 for CRST×L-EV). For the comparison of CON+veh, CRST+veh, CRST+L-EV, and CRST+IMI groups, one-way ANOVA followed by Newman-Keuls post hoc test was used (Ngf, F(3, 14)=1.495 and p=0.2591; tBdnf, F(3, 14)=2.938 and p=0.0699; Bdnf1, F(3, 11)=14.81 and p=0.0004; Bdnf4, F(3, 13)=5.176 and p=0.0143; Nt3, F(3, 12)=9.203 and p=0.0020; Nt4/5, F(3, 11)=2.170 and p=0.1493; Trkb, F(3, 14)=3.280 and p=0.0527). (D) Western blot analysis of proBDNF and BDNF expression in the hippocampus of CON+veh, CON+L-EV, CRST+veh, and CRST+L-EV (n=8 animals and 3~5 WB repeats per group). Two-way ANOVA followed by Bonferroni post hoc test (proBDNF, F(1,16)=22.62 and p=0.0002 for CRST main effect, F(1,16)=4.830 and p=0.0430 for L-EV main effect, and F(1,16)=3.681 and p=0.0731 for CRST×L-EV; BDNF, F(1,8)=1.329 and p=0.2823 for CRST main effect, F(1,8)=10.25 and p=0.0126 for L-EV main effect, and F(1,8)=0.1521 and p=0.7067 for CRST×L-EV). (E~H) Behavior tests (#1) on post-stress days 14~16. Representative tracking of mice placed in the sociability test (E). The percent time (%) spent in the target or non-target fields (F), and immobility time in the TST (G) and FST (H) of CON+veh, CON+L-EV, CRST+veh, CRST+L-EV, and CRST+IMI (n=6~12 animals per group). Two-way ANOVA followed by Bonferroni post hoc test was used for the comparison of CON+veh, CON+L-EV, CRST+veh, CRST+L-EV (target field in the sociability test, F(1,27)=2.243 and p=0.1458 for CRST main effect, F(1,27)=0.6705 and p=0.4200 for L-EV main effect, and F(1,27)=9.550 and p=0.0046 for CRST×L-EV; non-target field in the sociability test, F(1,27)=2.420 and p=0.1315 for CRST main effect, F(1,27)=0.5370 and p=0.4700 for L-EV main effect, and F(1,27)=9.869 and p=0.0041 for CRST×L-EV; TST, F(1,34)=7.324 and p=0.0.106 for CRST main effect, F(1,34)=0.6786 and p=0.4158 for L-EV main effect, and F(1,34)=5.308 and p=0.0275 for CRST×L-EV; FST, F(1,35)=29.00 and p<0.0001 for CRST main effect, F(1,35)=5.846 and p=0.0210 for L-EV main effect, and F(1,35)=18.19 and p=0.0001 for CRST×L-EV). One-way ANOVA followed by Neuman-Keuls post hoc test was for the comparison of CON+veh, CRST+veh, CRST+L-EV and CRST+IMI (target field in the sociability test, F(3,27)=6.470 and p=0.0019; non-target field in the sociability test, F(3,27)=6.435 and p=0.0020; TST, F(3,33)=6.772 and p=0.001; FST, F(3,34)=21.57 and p<0.0001). (I~L) Behavior tests (#2) on post-stress days 28~30. Representative tracking of mice placed in the sociability test (I). The percent time (%) spent in the target or non-target fields (J), and immobility time in the TST (K) and FST (L) of CON+veh, CON+L-EV, CRST+veh, CRST+L-EV, and CRST+IMI (n=6~12 animals per group). Two-way ANOVA followed by Bonferroni post hoc test was used for the comparison of CON+veh, CON+L-EV, CRST+veh, CRST+L-EV (target field in the sociability test, F(1,28)=1.580 and p=0.2192 for CRST main effect, F(1,28)=9.744 and p=0.0041 for L-EV main effect, and F(1,28)=1.332 and p=0.2582 for CRST×L-EV; non-target field in the sociability test, F(1,28)=1.557 and p=0.2224 for CRST main effect, F(1,28)=9.706 and p=0.0042 for L-EV main effect, and F(1,28)=1.304 and p=0.2632 for CRST×L-EV; TST, F(1,36)=2.902 and p=0.0971 for CRST main effect, F(1,36)=0.6042 and p=0.4421 for L-EV main effect, and F(1,36)=6.737 and p=0.0136 for CRST×L-EV; FST, F(1,35)=6.714 and p=0.0139 for CRST main effect, F(1,35)=5.713 and p=0.0224 for L-EV main effect, and F(1,35)=5.783 and p=0.0216 for CRST×L-EV). One-way ANOVA followed by Neuman-Keuls post hoc test was for the comparison of CON+veh, CRST+veh, CRST+L-EV and CRST+IMI (target field in the sociability test, F(3,27)=4.567 and p=0.0103; non-target field in sociability test, F(3,27)=4.535 and p=0.0106; TST, F(3,31)=6.029 and p=0.0023; FST, F(3,34)=5.762 and p=0.0027). Data are presented as mean±SEM. *p<0.05; **p<0.01.
© Exp Neurobiol
